Polyunsaturated fatty acids (PUFA) refers to a strait chain fatty acid having two or more double bonds and a carbon chain with a length of 16-22 carbon atoms, which are important components of cell membranes and biofilms in an organism and able to regulate cell configuration, dynamic equilibrium and maintain cell membrane relative liquidity, thereby maintaining normal physiological functions of a cell. Therefor they can influence cell chemical composition and functions such as signal transmission, immunity and the like, and thus have great contribution to occurrences of relative disease. PUFA possesses important physiological regulation functions in human body, comprising esterifying cholesterol, reducing cholesterol and triglyceride in blood, reducing blood viscosity, improving blood microcirculation, increasing activity of brain cells and enhancing memory and intelligence, etc. PUFA includes eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and the like, wherein DHA can prevent and treat cardiovascular disease, improve blood viscosity and increase deformability of erythrocytes, prevent and treat cancer, reduce thrombus and inflammation. Also, DHA is an main component of human brain, playing an important role in normal brain growth of infants and on normal functioning of adult brains. Some research suggests that the lack of DHA leads to decrease brain function, so, experts recommend that adults and pregnant women should supply with PUFA-rich food, such as marine fish for moderate supplement. DHA is a commendable component for medicines and nutrients.
Considering the important functions and effects of PUFA, it is widely focused in medicine and food fields. The conventional preparation of PUFA is separating from fish oil. However, the PUFA separated from fish oil has a strong fishy taste, a limited source and an unstable yield. The amount of W-3 unsaturated fatty acid in fish oil varies depending on fish spices as well as fishing seasons, weather and locations, and the production of fish oil has fluctuated output and involves complicated purification processes and the product yield is low. Meanwhile, the occurrence of reckless overfishing for the commercial profits, because of the big demand, brings bad effects on environmental resource protection. Therefore, the conventional preparation of fish oil cannot meet the needs.
Currently, it has been found that microorganism can be used for producing PUFA and plenty of research on it has been done. Particularly the method for producing PUFA such as DHA and the like by fermentation of algae has been the current research focus. This method goes beyond the limitation of conditions such as season, location and the like, obtains fatty acid with high content and simple component, greatly simplifies the purification process, decreases the difficulty of refining, and the culture condition thereof is easier to control, which provides the possibility of controlling the amount and constituent of lipid. Producing PUFA such as DHA by fermentation of microorganism also can reduce the negative effect on environmental resource caused by excessively fishing because of market demand.
The method for producing PUFA by fermentation of microorganism can realize the operable control of temperature, dissolved oxygen, pH value, thereby easier to realize culture process and the controllability and stability of products. Due to the appearance of metabolic products and/or the consumption of some component in the medium, the common problem during the fermentation is a drastic undulation of pH value. In this condition, if pH value is not suitably regulated during the fermentation of microorganism, the metabolic rate and cell growth will be impacted, and even the cell growth stops. Accordingly, a regulation of pH value is needed in fermentation of microorganism.
In the prior art, the conventional method for pH value regulation during the process for producing PUFA by fermentation of microorganism is to supply an acid-base solution to the fermentation system with an external device. Although this method can control pH value in a stable range to some degree, there still exists come problems, such as extra devices, sterilization process and labor costs, and more worse, an increased risk of microorganism contaminations during the fermentation process, for once there occurs microorganism contamination during the fermentation, it will have a dramatic effect on stable production and cause a great economic loss.
Beside the use of an external device, in recent years, adjusting the constituent of culture medium so that pH value thereof can be regulated by itself also is a research area and an effective mean to regulate pH value during the fermentation process, comprising:
(1) Adding strong alkali-weak acid salts to medium. CN101538592B discloses a method for producing docosahexaenoic by industrial Crypthecodinium cohnii fermentation, wherein sodium glutamate is added to the medium thereby omitting a pH value regulation during fermentation; CN101519676B discloses a method for producing docosahexaenoic by industrial Crypthecodinium cohnii fermentation, wherein sodium glutamate is added to the medium and citric acid is supplied to the system during fermentation; CN1218035C discloses a method for producing long chain PUFA with heterotrophic culture of marine microalgae, wherein sodium glutamate is added to the medium thereby omitting a pH value regulation during fermentation; CN101591617B discloses a strain producing docosahexaenoic and the mutation screening and use thereof, wherein sodium glutamate and sodium bicarbonate are added to the medium thereby omitting a pH value regulation during fermentation. In these methods comprising supplying strong alkali-weak acid salts for example sodium glutamate as nitrogen source in the medium, the pH value has a notable increase during the fermentation process as strong alkali-weak acid salts are consumed by the cell. As excessively high pH value will severely affect and change the metabolic process in cell, thus causing a decreased yield in fermentation process. However, when an external device is needed to supply the acid solution for pH value regulation, it not only increases the equipment investment, but also takes a risk of contaminations.
(2) Adding strong acid weak alkali salts such as inorganic ammonium salts to medium. CN101528939B discloses using an improved medium to produce ω-3 fatty acid from the Thraustochytriales, wherein sodium hydroxide is supplied during fermentation for pH value regulation; CN101812484B discloses a method for producing DHA via fermentation by high density Schizochytrium culture wherein ammonium hydroxide is supplied to the system for pH value regulation. A series of inventions by Richard B. Bailey et al respectively disclose methods for increasing the generation of lipid containing polyenoid fatty acids by high density eukaryotic microorganism culture in a fermentor and ammonium hydroxide is supplied in fermentation process for pH value regulation. In these methods comprising supplying strong acid-weak alkali salts like inorganic ammonium salts as nitrogen source in the medium, the pH value has a notable decrease during the fermentation process as strong acid-weak alkali salts are consumed. Excessively low pH value results in rancidity in fermentation system, which will severely affect and change the metabolic process in cell, thus causing a decreased yield in fermentation process. Meanwhile strong acid circumstance does seriously harm to fermentation devices, which makes it unsuitable to constantly and stably produce unsaturated fatty acids with microorganism. If supplying a base solution or other one with high pH value for pH value regulation, it not only increases the equipment investment, but also takes a risk of contaminations.
In addition, CN1914327A discloses a method for THRAUSTOCHYTRIALES microorganism culture, which regulates and stabilizes pH value in fermentation process by adding calcium carbonate to the medium. Due to limited solubility in water carbon dioxide generated from calcium carbonate during fermentation process, however, the buffer capacity of the buffer system decreases during fermentation process.
Accordingly a novel solution to medium is required to realize highly effective and stable control on the pH value during fermentation process in the art.